This invention relates to a novel .gamma.-L-glutamyl-4-nitroanilide derivative which is useful as a substrate for determining .gamma.-glutamyl transpeptidase (hereinafter abbreviated as .gamma.-GTP) activity, and a process for determining .gamma.-GTP activity using the same.
.gamma.-GTP is a membrane-bound enzyme which has activity to hydrolyze .gamma.-glutamylpeptide to transfer the .gamma.-glutamyl group to other peptides or amino acids.
Determination of .gamma.-GTP activity is widely utilized, for example, for diagnosis of liver and file duct diseases and screening of alcoholism. Recently, a .gamma.-GTP isozyme which appears specifically in serum of a hepatoma patient has been found, and the relationship between .gamma.-GTP in urine and pathosis has been reported. Thus, the diagnostic significance of determination of .gamma.-GTP activity is noted again.
As a process for determining .gamma.-GTP activity, various processes have so far been proposed and made practicable, and a rate assay method using .gamma.-L-glutamyl-p-nitroanilide as a substrate is the most usual and is now often employed.
However, .gamma.-L-glutamyl-p-nitroanilide as a substrate is disadvantageous in that it is slightly soluble in a suitable pH range for determining .gamma.-GTP activity and is unstable in such a suitable pH range for dissolution.
On the other hand, as water-soluble substrates free from these disadvantages, there are .gamma.-L-glutamyl-3-carboxy-4-nitroanilide, .gamma.-L-glutamyl-3-sulfo-4-nitroanilide, etc. (the specifications of U.S. Pat. Nos. 3, 979,447, 3,986,931 and 4,049,702). These water-soluble substrates are much superior to .gamma.-glutamyl-p-nitroanilide in both solubility and stability after dissolution, but they are not always sufficient in stability after dissolution. Thus, their improvement has been desired.
In determination of .gamma.-GTP activity in human serum, serum containing a known concentration of .gamma.-GTP (control serum) is often used as a standard or a maker for quality control. As the .gamma.-GTP added to such control serum, those derived from bovine kidney and porline kidney are mainly used. There is a high possibility of the presence of serum hepatitis virus in human serum having a high .gamma.-GTP activity, and so purification of .gamma.-GTP from the human serum is difficult from a hygienic viewpoint. A human tissue containing a large amount of .gamma.-GTP is not easily available. For these reasons, .gamma.-GTPs derived from bovine kidney and procine kidney are used in place of human serum .gamma.-GTP. However, such water-soluble substrates as described above have a more strongly polar group (a water-soluble group) than does .gamma.-L-glutamyl-p-nitroanilide. Therefore, they tend to vary in reactivity as substrates, depending on the electrically charged state of .gamma.-GTP particularly in the vicinity of the active site, and they vary considerably in reactivity with .gamma.-GTP as substrates, depending on the source of .gamma.-GTP such as human liver, bovine kidney, porcine kidney, etc. That is to say, when the activity value (international unit (IU)/l) of .gamma.-GTP in a sample prepared by dissolving .gamma.-GTP derived from each of the above-mentioned various sources in a predetermined concentration (M) is measured by use of the abovementioned water-soluble substrate, the difference among the activity values thus measured is considerably large. Therefore, in the case of determining .gamma.-GTP in human serum by use of a control serum containing .gamma.-GTP derived from bovine kidney and/or porcine kidney as a standard or a maker for quality control, employment of the above-mentioned water-soluble substrate has been disadvantageous in that the control serum should be controlled more strictly, as compared with a conventional determination method using .gamma.-L-glutamyl-p-nitroanilide as a substrate.